Use of sirnas for gene silencing in antigen presenting cells

ABSTRACT

The present invention relates to the use of small interfering RNAs (siRNAs) for silencing gene expression in antigen-presenting cells such as dendritic cells, in particular for immunomodulatory purposes.

The present invention relates to the use of small interfering RNAs(siRNAs) for silencing gene expression in antigen-presenting cells suchas dendritic cells, in particular for immunomodulatory purposes.

RNA interference (RNAi) is a mechanism involving double-stranded RNA(dsRNA) molecules and resulting in post-transcriptionalsequence-specific silencing of gene expression.

It is a multistep process, involving in a first step the cleavage,through the action of the Dicer enzyme (a RNase III endonuclease), oflarge dsRNAs into 21-23 ribonucleotides-long double stranded effectormolecules called small interfering RNAs (siRNAs). These siRNAs duplexesbind to a protein complex to form the RNA-induced silencing complex(RISC). The RISC specifically recognises and cleaves the endogenousmRNAs containing a sequence complementary to one of the siRNA strands.

This mechanism was initially described in plants, worms, drosophila andparasites, where dsRNAs have been successfully used to inducegene-specific post transcriptional silencing.

However, in upper animals, such as vertebrates and in particularmammals, large dsRNAs (longer than 30 bp) elicit a type I interferonresponse predominantly leading to the activation of protein kinase R(PKR) (WILLIAMS, Oncogene 18, 6112-6120, 1999). In many cell types, thisresults generally in a nonspecific degradation of RNA transcripts and ageneral shutdown of translation.

This obstacle to the use of RNA interference for gene specific silencingin mammals has been recently overcome by the use of siRNAs (TUSCHL etal., Genes Dev. 13, 3191-3197, 1999; ELBASHIR et al., Nature 411,494-498, 2001). By way of example, siRNAs consisting of 19-25,preferably 19-23 nucleotides, with overhanging 3′-ends are described inPCT WO 02/44321.

Due to their small size, the siRNAs fail to activate the PKR pathway,and it has been shown that they were able to induce a specific andstrong reduction of protein expression in cultures of fibroblast andepithelial cell lines (HARBORTH et al., J. Cell. Sci. 114, 4557-4565,2001), and of primary lymphocytes (JACQUE et al., Nature 418, 435-438,2002) as well as in vivo in mice (McCAFFREY et al., Nature, 418, 38-39,2002).

Antigen presenting cells (APC) constitute a complex system of cells thatcapture, process and present antigens to lymphocytes and play prominentroles in infectious diseases, cancer, immune disorders and vaccination.APCs include monocytes/macrophages, B lymphocytes, dendritic cells (DC);the most potent APCs being DC. The DC system consists of a complexsystem of cells that are uniquely capable of activating naive Tlymphocytes thus, unlike other APCs, can initiate immune responses. Awell-characterized type of DC is the monocyte-derived DC that isproduced in vitro by culture of human blood monocytes.

There is great interest in understanding mechanisms of DC activation. DCintegrate a variety of signals from pathogens, inflammatory mediators orT cells that condition their ability to present antigen to naive T cellsand to subsequently regulate the development of immune responses(LANZAVECCHIA et al., Cell 106, 263-266, 2001 MELLMAN et al., Cell 106,255-258, 2001). One can recognize three major categories of signals thatregulate the function and activation of DC. The first relates to therecognition and processing of pathogens or antigen-associated motifs.Bacterial and viral constituents such as lipopolysaccharides (LPS),dsRNA, CpG motifs of bacterial DNA are recognized by specializedToll-like receptors (TLR) on DC and trigger cytokine production andcellular activation of DC. Another influence on DC is the environmentalmilieu for instance cytokines, chemokines, hormones or small moleculesthat have pro- or anti-inflammatory activity and are produced duringinnate or adaptive immune responses. Notably, interleukins (IL) likeIL-1 or IL-4 modulate the differentiation of DC and their response toother activation signals. A third type of signal involves receptors andligands engaged by cognate cell-to-cell interactions. Examples includeinteractions between DC and T lymphocytes via molecules of the tumornecrosis factor (TNF) receptor/ligand superfamilies that are prominentregulators of DC activation, survival and differentiation. For example,CD40 ligand (CD40L), induces the maturation of DC in vitro, enhancingtheir ability to interact with naive T cells through up-regulation ofMHC class II and co-stimulatory antigens on the cell surface. Further,CD40L in conjunction with mediators of innate immunity such as IL-1,induces the transcription of IL-12α and β mRNA and the production ofhigh levels of the heterodimer interleukin-12 (IL-12)αβ in DC (WESA &GALY, Int. Immunol., 2001, August; 13, 1053-61; LUFT et al., J. Immunol.168, 713-722, 2002). The cytokine IL-12 is a deterministic factor forthe development of cellular immunity, inducing Th1 T celldifferentiation and the production of high levels of IFN-γ by T andNatural Killer (NK) lymphocytes (TRINCHIERI et al., Curr. Top.Microbiol. Immunol. 238, 57-78, 1999).

Thus, it appears that the molecular mechanisms that regulate DCactivation and the production of cytokines by DC are pivotal events thatcontrol the development of cellular immune responses.

The transduction of signals from TNF receptor superfamily and theinterleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily ismediated by TNF receptor associated factors (TRAFs). To date, sixmembers of this family of homologous proteins have been described. TRAFproteins are important regulators of cell death, cellular responses tostress and TRAF2, TRAF5 and TRAF6 have been reported to mediateactivation of NF-kappaB and jun kinase. In DC, TRAF-3 is recruited inmembrane rafts by engagement of CD40 on the surface of the DC (VIDALAINet al., EMBO J. 19, 3304-3313, 2000). Thus potentially, TRAF-3 plays animportant role in the response of DC to this mode of activation but arole for TRAF-3 in DC has not been clearly established. Mice renderedgenetically null for TRAF3 die rapidly and fail to develop a competentimmune system (XU et al., Immunity 5(5), 407-415, 1996).

In DC, pro-inflammatory signals of innate or adaptive immune responsesgenerally lead to the activation of NF kappa B/Rel for the transcriptionof target genes. In mammalian cells, NE kappa B/Rel proteins consist ofp50 (NF-κB1), p52 (NF-κB2), p65 (RelA), RelB, c-Rel that are encoded bydifferent genes and play non-redundant roles of importance in variousaspects of development, inflammation and immunity (BURKLY et al., Nature373, 531-536, 1995; FRANZOSO et al., J. Exp. Med. 187, 147-159, 1998).NF kappa B/Rel proteins form homo- or hetero-dimers maintained in thecytosol by association to inhibitory IKB proteins. A variety ofinflammatory, pathogen-derived, stress or developmental stimuli,transmitted by the pathways mentioned above, activate the IKB kinasecomplex, subsequently triggering the phosphorylation of IKB and itsdegradation in the proteasome. This releases p50 or p52 that form, withRel proteins, heterodimers that are translocated to the nucleus andactivate the transcription of target genes (GHOSH et al., Annu. Rev.Immunol. 16, 225-260, 1998). Further phosphorylation events regulate theactivity of Rel proteins in the nucleus. In addition, homodimers of p50or p52 exist that acquire transactivating potential by binding to Bcl-3,a member of the IKB family of proteins.

A major role of NF kappa B/Rel proteins in antigen presentation has beenfirst suggested by localization studies in tissues or in cells then bythe phenotype of animals with targeted mutations. Individually, p50,IκB-α, c-Rel, RelB, p65, Bcl-3 and p52 knockout mice have been producedwith impairment of several immunologic parameters (reviewed in SHA, J.Exp. Med. 187, 143-146, 1998). OUAAZ et al. (Immunity. 16, 257-270,2002) report that development and function of murine BM-derived DC werenot affected by lack of individual NF kappa B subunits, while on theother hand the combined absence of p50 and Rel-A abrogates the formationof all subsets of DC; the lack of p50 and c-Rel together stronglyreduced IL-12 production but had no significant effect on expression ofMHC and costimulatory molecules. In human cells, differential expressionof NF kappa B/Rel genes is found during the in vitro differentiation ofmonocytes into DC or macrophages and complexes consisting of p50, RelBand c-Rel are found in the nucleus of mature monocyte-derived DC(RESCIGNO et al., J. Exp. Med. 1188, 2175-2180, 1998; NEUMANN M et al.,Blood. 95, 277-285, 2000). Transfection of RelB cDNA in B cell linesincreases expression of MHC class I and CD40 cell surface expression andenhances MHC class I-peptide-mediated activation of CD8⁺ T cells(O'SULLIVAN et al., Proc Natl Acad Sci USA. 97, 11421-11426, 2000).Thus, NF kappaB/Rel proteins are associated with the development of theantigen-presenting cell system as their expression correlates with theactivation of various types of APCs and with the differentiation ofnon-professional APCs such as monocytes/macrophages into professionalAPCs like dendritic cells. However, it is unclear how individualconstituents of NF kappa B regulate the activation of human DC.

As a viral constituent, dsRNA is recognized by APCs as apathogen-associated motif that leads to cellular activation. Thus,dendritic cells react to stimulation with dsRNA in a quite different waythan other cell types: in contrast to other cells where dsRNA inducesvia the activation of PKR a general shutdown of translation, dendriticcells respond to dsRNA by an increase in protein synthesis, andup-regulation of MHC and co-stimulatory antigens, allowing a high levelof production and presentation of viral antigens (CELLA et al., J. Exp.Med. 89(5), 821-829, 1999). It has been reported (ALEXOPOULOU et al.,Nature 18, 413, 732-738, 2001) that DC specifically recognize dsRNA viaToll-like receptors, in particular Toll-like receptor 3 (TLR3);activation of this receptor induces the activation of NF-κB and theproduction of type I interferons. Messenger RNA for TLR3 has been foundin immature and mature monocyte-derived DC but its presence in monocytesis controversial (VISINTIN et al., J. Immunol. 166, 249-255, 2001;KADOWAKI et al., J. Exp. Med. 17, 194(6), 863-869, 2001). Collectively,the expression of TLR is not restricted to antigen-presenting cells butis found also on leukocytes and fibroblasts. However, only DC expressthe full repertoire of TLR, in particular, DC are the only leukocytesthat express TLR3, the putative receptor for dsRNA. (MUZIO et al., J.Immunol. 164, 5998-6004, 2000). It has been shown that binding of dsRNAto DC or to TLR3-transfected epithelial cells induces an IFN response(KADOWAKI et al., precited; MATSUMOTO et al., Biochem. Biophys. Res.Commun., 293, 1364-1369, 2002). These results suggest that the uptakeof, and response to, dsRNA may be distinct in DC expressing TLR3 such asmonocyte-derived DC, compared to other types of cells: these resultsalso suggest that the potential activation of TLR3 by siRNA could causenon-specific IFN response, mortality and translation shut-down, thuspreventing the effective use of siRNA in DC.

In view of the above, the functionality of RNA interference in APC wasuncertain, since a mechanism resulting in elimination of viral RNA wouldresult in a decrease in the production of viral antigens, and thus in aless efficient presentation thereof.

The inventors have tested if siRNAs were able to induce in dendriticcells either a non-specific type-I interferon response or a genespecific silencing.

They have found that double stranded RNA molecules of 21-23ribonucleotides did not elicit any non-specific type-I interferonresponse. In contrast, they found that a strong gene specific silencingwas elicited when these RNA molecules were siRNAs directed against genesexpressed in dendritic cells.

In particular, they found that the transfection of dendritic cells withsiRNA directed against the p50 gene induced a specific decrease of p50expression. In contrast with the observations previously reported byOUAAZ et al., they found that this reduction of p50 expression wassufficient to induce a strong reduction of secretion of IL-12, and thatco-transfection of DC with siRNA directed against the p50 gene and siRNAdirected against the c-Rel gene further induced a significant reductionof the expression of MHC and costimulatory molecules.

In addition, they found that transfection of dendritic cells with siRNAdirected against the gene encoding TNF-receptor associated factor 3induced a strong reduction of secretion of IL-12.

Further, they also found that DC transfected with siRNA directed againstgenes encoding either p50 or TRAF3 failed to activate the production ofIFN-gamma by T lymphocytes.

The invention thus provides new means for modulating the immuneresponse, through siRNA mediated gene silencing in dendritic cells, morespecifically human dendritic cells. In particular, the inventionprovides means for decreasing IL-12 production by dendritic cells. Theinvention also provides means for suppressing an unwanted Th1 T cellresponse.

The present invention thus relates to the use of siRNAs to down-regulatethe expression of one or more target(s) gene(s) in an antigen presentingcell, in particular a dendritic cell or a precursor thereof, andpreferably a monocyte-derived dendritic cell or a precursor thereof.Advantageously, said antigen presenting cell is a human cell.

In particular, an object of the invention is a method for obtainingisolated or cultured antigen presenting cells wherein the expression ofone or more target(s) gene(s) is down-regulated, wherein said methodcomprises introducing in said cells siRNA(s) directed against saidtarget(s) gene(s).

From the sequence of a chosen target gene, one of skill in the art caneasily design and prepare siRNA directed against said target gene, bymeans known in themselves, as disclosed for instance by ELBASHIR et al.,(Nature, 2001, cited above; EMBO J. 20, 6877-6888, 2001) or in PCT WO02/44321. Introduction of said siRNA in the cells can be performedeither by direct transfection, for instance by electroporation orliposome mediated transfection, or by means of an expression vectorcomprising a DNA template for the chosen siRNA placed undertranscriptional control of a polIII promoter. A DNA template for siRNAcomprises the DNA sequences to be transcribed into the sense andantisense strands constituting the siRNA duplex. At the present time,two kinds of expression vectors for siRNA have been proposed (TUSCHL,Nature Biotechnol., 20, 446-448, 2002). In the first one, the sense andantisense sequences of the DNA template are placed in separatetranscription units (LEE et al., Nat. Biotechnol. 20, 500-505, 2002;MIYAGISHI & TAIRA, Nat. Biotechnol., 20, 497-500, 2002). In the secondone, a single promoter controls the expression of the sense andantisense sequences of the DNA template, that are separated by a shortspacer region; the transcription of this construct results insmall-hairpin RNA (shRNA) that give rise to siRNA after intracellularprocessing involving the enzyme Dicer (McCAFFREY et al., Nature, 2002,cited above; BRUMMELKAMP et al., Science, 296, 550-553, 2002; PADDISONet al., Genes Dev. 16, 948-958, 2002).

A particular embodiment of the invention includes the selection of atarget gene among:

-   -   a gene encoding the p50 subunit of NF-κB;    -   a gene encoding TNF-receptor associated factor 3;    -   a gene encoding the c-Rel subunit of NF-κB.

Another embodiment of the invention includes the selection of a targetgene encoding the p50 subunit of NF-κB and a target gene encoding thec-Rel subunit of NF-κB.

The invention also encompasses siRNA directed against a target geneselected among:

-   -   a gene encoding the p50 subunit of NF-κB;    -   a gene encoding TNF-receptor associated factor 3;    -   a gene encoding the c-Rel subunit of NF-κB; as well as        expression vectors comprising a DNA template for said siRNA.

Expression vectors of the invention include gene therapy vectors, inparticular gene therapy vectors derived from viruses such as MurineMoloney Leukemia virus, Human immunodeficiency virus (HIV-1), Simianimmunodeficiency virus (SIV), foamy virus, adeno-associated virus,adenovirus, canine adenovirus, canarypox virus, herpes virus. Preferredvirus-derived vectors for antigen presenting cells, including dendriticcells, are derived from Murine Moloney Leukemia virus, HIV, SIV, oradenovirus.

Another object of the invention is the use of siRNAs or expressionvectors of the invention as medicaments.

According to a preferred embodiment of the invention, siRNA directedagainst a target gene selected among:

-   -   a gene encoding the p50 subunit of NF-κB;    -   a gene encoding TNF-receptor associated factor 3;    -   a gene encoding the c-Rel subunit of NF-κB;

or a vector expressing said siRNA is used for preparing a therapeuticcomposition, in particular an immunosuppressive composition, fortreating or preventing a disease resulting from an overproduction ofIL-12 by dendritic cells.

Diseases resulting from an overproduction of IL-12 by dendritic cellsinclude for instance pathologic conditions in which adaptive responsesare elicited against self-antigens, such as autoimmune diseases rangingfrom systemic to organ specific such as systemic lupus erythematosus,rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetesmellitus, Hashimoto's thyroiditis, myasthenia gravis.

An overproduction of IL-12 is also implied in adverse immune responseagainst the graft in tissue or organ transplantation, or against vectorsused to correct genetic deficiencies in gene transfer therapies.Accordingly, the siRNAs of the invention, or the correspondingexpression vectors, can also be used in the treatment of diseasesresulting from said immune response. In some cases, one may wish toobtain a more drastic immunosuppressive effect: this can be done byreducing at once the production of IL-12 and the expression of MHC andcostimulatory molecules, by use of a combination of siRNA directedagainst a target gene encoding the p50 subunit of NF-κB, with siRNAdirected against a target gene encoding the c-Rel subunit of NF-κB, orof the corresponding expression vectors.

The present invention also provides antigen presenting cells, inparticular dendritic cells or precursors thereof, obtained by the methodof the invention. These antigen presenting cells contain siRNA(s)directed against target gene(s) expressed in said dendritic cell.

The invention further provides pharmaceutical compositions comprisingantigen presenting cells of the invention. The invention also providespharmaceutical compositions comprising T lymphocytes and dendriticcells.

The present invention also provides a method to produce T lymphocytesthat fail to produce IFN-gamma, wherein said method comprises inducingthe activation of naïve T cells by co-cultivating said T cells with anantigen presenting cells of the invention, containing siRNA directedagainst a gene encoding p50 or TRAF-3.

The present invention will be further illustrated by the additionaldescription which follows, which refers to examples demonstrating theeffect of siRNAs in dendritic cells. It should be understood howeverthat these examples are given only by way of illustration of theinvention and do not constitute in any way a limitation thereof.

EXAMPLE 1 EFFECT OF SIRNA TARGETING NF KAPPA B P50 AND C-REL INDENDRITIC CELLS

siRNAs

21-nucleotide double-stranded RNA with two overhangs dT nucleotides,targeting NFκB p50 (GGG GCU AUA AUC CUG GAC UdTdT; SEQ ID NO:1), andcRel (CAA CCG AAC AUA CCC UUC U dTdT; SEQ ID NO:2) were designed fromthe sequences of the corresponding genes. Control double-stranded RNAshaving randomly scrambled sequences (scramble I: UGU UUU AAG GGC CCC CCGUdTdT; SEQ ID NO:3, scramble II: CGG CAG CUA GCG ACG CCA UdTdT; SEQ IDNO:4) were also prepared.

The sequences indicated above are the sense sequences of the siRNAs. Thesequence for p50 as well as the sequence for cRel failed to revealsignificant sequence homologies with other known genes (including othermembers of the same families) after standard BLAST search. Similarly,control scramble RNAs failed to reveal significant sequence homologieswith any known genes after standard BLAST search.

Dendritic Cells

Mononuclear cells (MNC) were isolated by centrifugation over Ficoll(Amersham Pharmacia Biotech, Piscataway, N.J.) (d<1.077 g/ml) from cordblood samples and were cryopreserved in liquid nitrogen using a 10% DMSOfreezing solution.

Monocytes were obtained by incubating MNC on tissue culture plates(2×10⁶ cells per ml per well in 24 well plates) in RPMI medium with 10%fetal bovine serum (FBS) (R10) ²⁷ in a humidified atmosphere at 37° C.,5% CO₂ for 2 hours, followed by washing to remove non-adherent cells.These adherent cells were cultured in R10 medium with GM-CSF (25 ng/ml,Immunex, Seattle, Wash.), and IL-4 (10 ng/ml, RD Systems, Minneapolis,Minn.) for 4 to 6 days to induce DC differentiation.

These immature human monocyte-derived DC cells were transfected byelectroporation with various concentrations of p50 or control siRNAs.

Transfection of siRNAs

Transfection of siRNAs was carried out by electroporation with a squarewave electroporation system (BTX ECM 830, San Diego, Calif.).

Briefly, 4×10⁵ cells in 0,4 gap cuvettes were subjected to 5 cycles of20V, 10 ms in electroporation buffer pH 7.6 (120 mM KCl, 0,15 mM CaCl2,10 mM K2HPO4/KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2, 50 mMGlutathion, 2 mM ATP).

Lack of Non-Specific Effect of siRNAs

Electroporation of DC did not induce significant toxicity in the cellsneither after transfer of scramble or p50 siRNAs. Less than 10% of thecells were dead as measured by Trypan blue exclusion in 7 experiments.

Since DC are particularly apt at recognizing pathogen motifs such asdouble stranded RNA via the expression of specific Toll-like receptors,it was first checked whether or not a type-I interferon response wasinduced after transfection of siRNA in DC.

Human Interferon α levels were determined using specific ELISA kit(Biosource International, Camarillo, Calif.). The lower limit ofdetection was 25 pg/ml.

The results are shown on FIG. 1. Furthermore, supernatant fluids from DCcultures that were transfected with siRNAs were added to cultures ofWISH fibroblasts infected with vesicular stomatatis virus and did notprevent the virus-induced lysis of WISH cells. This bio-assay furtherconfirms the lack of type-I interferon production in culture mediumafter siRNA transfection of DC.

These results show that neither control siRNA nor p50 siRNA inducesdetectable type-1 IFN production.

Down-Regulation of p50

48 hours after electroporation with varying doses (1, 10, 50, 100, or150 nM) of scramble or p50siRNA, expression of p50 in DC was evaluatedby immuno-fluorescence.

0.5-1×10⁵ dendritic cells were spun on coverslips and fixed with 4%paraformaldehyde during 10 min at 4° C. Cells were washed twice in PBSthen permeabilized in saponin buffer (0.1% saponin, 0.2% BSA, 0.02%sodium azide, in PBS). Non-specific Fc binding was blocked by incubationfor 10 min. on ice with excess human gamma-globulin (1 mg/ml) and 1/100dilution of donkey serum (Sigma, Saint Quentin Fallavier, France).Polyclonal goat antibodies specific for NFkB p50 (Sc-1191) (Santa CruzBiotechnologies, Santa Cruz, Calif.) were used at 5 μg/ml followed by aFITC conjugated donkey anti-goat secondary reagent (JacksonImmunoresearch, West Grove, Pa.) used at 1/400 dilution in saponinbuffer. Cells were observed under epifluorescence microscopy.

A dose-dependent extinction of p50 is particularly visible in thenucleus of DC with as little as 10 nM of p50 siRNA.

Results, expressed as percent of nucleated cells in the preparationwhose nuclei show a dose-dependent extinction of p50 afterelectroporation with varying doses of control or p50 siRNA, are shown onFIG. 2. A significant down-regulation of p50 was obtained with 50 nM ofp50 siRNA. The extinction was optimal with 100 nM siRNA (overallapproximately 50% extinction; data not shown). Electroporation with 150nM siRNA did not induce a significant increase of the extinction.

In order to confirm these results, the expression of the p50 protein andthe p50 mRNA in DC electroporated with 100 nM of scramble or p50siRNAwere respectively analyzed by Western blot and RT-PCR. Western blot.

After electroporation, 5×10⁵ cells were spun, resuspended in lysisbuffer (50 mM tris, 150 mM NaCl, 1% TritonX100, 1% sodium Deoxicholate,0.1% SDS, 5 mM EDTA, protease inhibitor cocktail) and kept at −80° C.until used. Equal amounts of protein (10 ug as determined by Bio-Rad DCProtein Assay, Bio-Rad, Hercules, Calif.) were separated on 10%polyacrylamide gels and transfert to nitrocellulose sheets. Polyclonalgoat antibodies specific of p50 (Sc-1191) were used at 1/100 dilution.Anti-β actine (Sigma) was used as internal control. Horse Peroxidaseconjugated rabbit anti-goat was used as secondary reagents at 1/5000dilution. Standart immunostainings were carried out using the ECLWestern Blotting Analysis System (Amersham Pharmacia, Buckinghamshire,England).

The results are shown on FIG. 3. Levels of p50 are specifically reducedby about half in DC transfected by p50 siRNA but not in untreated cellsor in cells transfected with control or irrelevant siRNAs. RT-PCRanalysis.

DC were electroporated with controls or p50 siRNA. After 24 hours, totalcytoplasmic RNA was extracted from 5×10⁵ sorted cells using TRIzolreagent (all reagents from Gibco-InVitrogen, Cergy Pontoise, France).RT-PCR was done to analyze expression of p50, c-Rel, p65 and β-actinegenes. PCR products were analyzed on 2% agarose gel electrophoresisstained with ethidium bromide.

The results are shown on FIG. 4. These results indicate that thereduction in p50 protein expression is due to a strong and specificdown-regulation of p50 mRNA levels.

Reduction of IL-12 Production by p50 siRNA.

Immature DC were transfected with anti-P50 or scramble siRNA. 48 h aftertransfection, cells were harvested and washed twice in cytokine-freemedium, prior to incubation with human recombinant CD40L trimer (1μg/ml; Immunex), IL-1β (10 ng/ml R&D Systems). After overnightactivation, supernatants were harvested and tested for IL-12 p70 byELISA, using the OptEIA ELISA set for IL-12p70, according tomanufacturer's instructions (BD-PharMingen). The lower limit ofdetection was 4 pg/ml.

The results of 3 independent experimentations are shown in table Ibelow. TABLE I Condition IL-12 p70 (pg/1000 cells) Exp #1 Non treated0.45 Scramble I siRNA 0.29 P50siRNA 0.015 Exp #2 Scramble II siRNA 0.447P50 siRNA 0.148 Exp #3 Scramble II siRNA 0.32 P50 siRNA 0.058

These results show that treatment of DC with a siRNA anti p50 prior toactivation with CD40L+IL-1 reproducibly and strongly reduces thesecretion of IL-12.

Effect of p50 and cRel siRNAs on DC Phenotypic Maturation

Mature DC acquire expression of CD83, high levels of costimulatoryantigens CD80 and CD86 and MHC class II molecules. To analyze thebiological consequences of p50 reduction in monocyte-derived DC,expression of cell surface markers after stimulation with CD40L+IL-1βwas measured by flow cytometric analysis on DC untreated or treated with150 nM of scramble siRNA or p50, cRel or p50+cRel siRNAs.

Stainings of surface molecules were performed with the followingantibodies: FITC conjugated mouse anti-human CDla, HLA-DR, PE conjugatedmouse anti-human CD80, anti-CD83, APC-conjugated mouse anti-humanHLA-DR, CD86. Cells were analyzed on a FACSCalibur instrument (BectonDickinson) and data were analyzed using WinMDI (Version 2.8) software.

It was observed that treatment with scramble, p50, or cRel siRNAsinduced no significant alteration in the expression of the maturationmarker CD83 or of co-stimulatory molecules CD80, CD86, CD40 or MHC classII antigens (FIG. 5). However, combination of p50 and c-Rel siRNAs had aprofound effect and reduced expression of HLA-DR, CD80 and CD86 on thecells with little effect on CD83 expression.

The results of treatments with p50, cRel or p50+cRel siRNAs on theexpression of HLA-DR and CD80 markers are shown on FIG. 5.

Effect of p50 siRNA on T Cell Stimulating Properties of DC

Monocyte-derived DC have strong T cell stimulating properties andamounts as low as 1-10% of cells in a T cell culture are known to induceT cell proliferation and secretion of IFN-γ.

A mixed leukocyte reaction (MLR) was used to test the immunologicproperties of DC transfected with p50 siRNA.

Purified T cells were prepared from cord blood mononuclear cells (MNC)using negative selection. MNC were incubated with human γ globulins (1mg/ml) to block non-specific Fc receptor binding, then with monoclonalantibodies (mAbs) purified from hybridomas obtained from ATCC (Manassas,Va.) and specific for glycophorin A (10F7MN), CD14 (3C10-1E12), CD32(IV3), CD11b (OKM1) and CD40 (G28-5). Red blood cells, phagocytes, Bcells, monocytes and CD4⁺ T cells were then removed using magnetic beadscoupled to goat anti-mouse antibodies (Dynal Inc., Lake Success, N.Y.).Magnetic bead selection was repeated after adding purified anti-CD20 andanti-HLA-DR antibodies (Caltag, Burlingame, Calif.) to further remove Bcell and APCs. The negative fraction routinely contained >95% CD3⁺ Tcells.

Allogeneic proliferation was performed by culturing for five dayspurified naive T cells (5×10⁴ cells per 0.2 ml of complete media perwell in triplicate) with allogeneic 30 h-transfected DC. During the last10 hours of culture, 1 μCi of (3H) thymidine (NEN, Boston, Mass.) wasadded to each well. Cells were harvested (Skatron Instruments, Maurepas,France) and counted using a liquid scintillation counter. Results areexpressed as cpm±SD of triplicate wells.

The results are shown in FIG. 6 A.

These results show that similar T cell proliferation is induced by nontreated DC (⋄), DC treated with p50 siRNA (◯) or DC treated withscramble I siRNA (□).

Interferon gamma (IFNγ) is a cytokine resulting from a Th1 polarizationof the immune response. It is produced by NK and T cells and itparticipates in the amplification of the immune response. In order tostudy qualitative aspects of the allogeneic response elicited, theproduction of IFNγ in the supernatants of the MLR was tested.

IFN-γ was measured using the OptEIA ELISA set for IFN-γ according tomanufacturer's instructions (BD-PharMingen). The lower limit ofdetection was 4 pg/ml.

The results are shown in FIG. 6 B. A strong reduction in IFN-γproduction is observed in cultures stimulated with DC treated with p50siRNA (◯) when compared to non treated DC (⋄) or DC treated withscramble I siRNA (□).

EXAMPLE 2 EFFECT OF siRNA TARGETING TRAF PROTEINS IN DENDRITIC CELLS

siRNAs targeting TRAF3 (GUG CCA CCU GGU GCU GUG CdTdT; SEQ ID NO:5) andTRAF2 (GAA UAC GAG AGC UGC CAC GdTdT; SEQ ID NO:6) were designed fromthe sequences of the corresponding genes.

The sequences indicated above are the sense sequences of the siRNAs. Thesequence for TRAF3 as well as the sequence for TRAF2 failed to revealsignificant sequence homologies with other known genes (including othermembers of the same families) after standard BLAST search. Controlscramble RNAs were also prepared, as described in Example 1.

Immature human monocyte-derived DC cells were transfected byelectroporation with 150 nM of TRAF3 or TRAF2 siRNA, as described inExample 1.

Transfected DC were tested for their capacity to produce IL-12 uponCD40L+IL-1 activation, as described in Example 1.

The results are shown in FIG. 7. While TRAF2 siRNA did not producesignificant effects, TRAF3 siRNA significantly reduced the IL-12p70production upon activation of DC.

A mixed leukocyte reaction (MLR) was used to test the immunologicproperties of DC transfected with TRAF3 siRNA. T cell activation andIFNγ production were measured as as described in Example 1.

As shown in FIG. 8, an important reduction in T cell proliferation wasobserved when DC were transfected with TRAF3 siRNA (◯). Only at veryhigh ratios of DC a little effect was observed with TRAF2 siRNAtransfected-DC (⋄) when compared to DC transfected with scramble I siRNA(□).

FIG. 9 shows that there is an important reduction in the production ofIFNγ by T cells stimulated with TRAF3 siRNA transfected-DC (◯), whencompared with DC transfected with TRAF2 siRNA (⋄) or scramble I (□)siRNAs.

EXAMPLE 3 CONSTRUCTION OF AN EXPRESSION VECTOR FOR A p50 siRNA

A plasmid comprising a DNA template for a p50 siRNA of SEQ ID NO:1 wasconstructed according to BRUMMELKAMP et al., (Science, 2002, citedabove). This plasmid comprise a hairpin consisting of the DNAcorresponding to the sense and antisense sequences of siRNA, separatedby a spacer loop. This hairpin is placed under transcriptional controlof the polIII promoter H1. Briefly, a sequence coding for the H1promoter was obtained by PCR from genomic DNA of human peripheral bloodmononuclear cells. This sequence was cloned into the EcoRI/HindIII siteof the pBluescript phagemid vector. A XhoI restriction site was createdby directed mutagenesis in position 5′ adjacent to the EcoR1 site, toobtain the pH1 plasmid. A BglII adapter sequence followed by the p50hairpin and by a HindIII adapter was cloned into the BglII/HindIII siteof the pH1 plasmid to obtain the pH1-shp50 vector, schematized on FIG.10.

The sequence of the region of interest between XhoI sites in thispH1-shp50-1 plasmid is as follows (SEQ ID NO:7):

GTCGACGGTATCGATAAGCTTTTCCAAAAAGGGGCTATAATCCTGGACTTCTCTTGAAAGTCCAGGATTATAGCCCCGGG GATCTGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCA GCGTTCGAATTCCTGCAG

Letters in bold indicate the P50 small hairpin sequence; lettersunderlined indicate the H1 promoter and letters in bold and italicindicate the XhoI cloning site.

1. A method for obtaining isolated or cultured antigen presenting cellswherein the expression of one or more target gene(s) is down-regulated,wherein said method comprises introducing in said cells siRNA(s)directed against said target gene(s).
 2. The method of claim 1, whereinsaid antigen presenting cells are dendritic cells or precursors thereof.3. The method of claim 1, wherein at least one of said target gene(s) isselected from the group consisting of: a gene encoding the p50 subunitof NF-κB; a gene encoding TNF-receptor associated factor 3; and a geneencoding the c-Rel subunit of NF-κB.
 4. A siRNA directed against atarget gene selected from the group consisting of: a gene encoding thep50 subunit of NF-κB; a gene encoding TNF-receptor associated factor 3;and a gene encoding the c-Rel subunit of NF-κB.
 5. An expression vectorcontaining a DNA template for a siRNA of claim
 4. 6. Animmunosuppressive therapeutic composition comprising a siRNA of claim 4.7. An antigen-presenting cell obtainable by the method of claim
 1. 8. Apharmaceutical composition comprising an antigen-presenting cell ofclaim
 7. 9. A method to produce T lymphocytes that fail to produceIFN-gamma, wherein said method comprises inducing activation of naïve Tcells by co-cultivating said T cells with antigen presenting cells ofclaim 7, containing siRNA directed against a gene encoding p50.
 10. Apharmaceutical composition comprising activated T lymphocytes obtainableby the method of claim
 1. 11. An immunosuppressive therapeuticcomposition comprising an expression vector of claim 5.